I'm prepping some NGS Illumina data for downstream analysis. To begin, I want to remove any sequencing/ligating adapters and multiplexing (barcoding) tags. To do this, I am using fastx_clipper, which is part of the FASTX-Toolkit. I've also using Trimmomatic for this in the past.

Here is my question... Both of these software packages only scan for a single orientation of the adapter you provide within the Illumina reads. However, I find many sequences in all orientations of the adapter, namely: forward, reverse, forward complement, reverse complement. In the forward orientation, the software detects and trims the adapter in >90% of the reads, but in the other 3 orientations the software only detects are trims adapters in ~5% of the reads.

So, is it possible for the adapters to be found in different orientations than the forward sense, or am I seeing artifacts of non-strict adapter matching? Do people usually trim adapters in every possible orientation? Any other suggestions for successfully handling adapters?

Seeing an adapter in the forward orientation is the result of a DNA fragment being shorter than the read length, it is a "normal" occurrence in these cases. The Illumina TrueSeq indexed sequencing adapters were designed in such a way that the same adapter sequence will be found on reads coming from both strands.

In that case adapters present in any other orientation most likely indicate a protocol failure, in which case probably the entire read should be removed.

@Istvan Thanks for the answer. This is what I expected, which makes me unsure of the FASTX results. Perhaps my stringency is simply too loose, only requiring 7 sequential adapter bases to be matched. I'll try a few variations and report back later.